Louis T.J. Delbaere

Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8 A resolution. A model for serine protease catalysis.

Abstract This paper describes the 1.8 A resolution structure of the microbial enzyme Streptomyces griseus protease A in its native conformation, and in complexes with Ac-Pro-Ala-Pro-Phe-OH (I), Ac-Pro-Ala-Pro-Tyr-OH (II) and Ac-Pro-Ala-ProPhe-H (IV), all at pH 4.1. Each of these structures has been extensively refined by restrained parameter least-squares. The agreement factors ( R = Σ∥F o ¦—¦F c ∥/Σ¦F 0 ¦ ) are 0.130, 0.133, 0.122 and 0.142, respectively. The resultant electron density maps show that the peptide aldehyde (IV) forms a covalent hemiacetal bond with Ser195, the refined distance from the carbonyl carbon of the aldehyde to Oγ of Ser195 being 1.73 A. The corresponding distances in the protease-peptide I and II complexes are 2.58 A and 2.66 A, respectively, but there is no continuous electron density from Oγ to these carbonyl carbon atoms. Only three regions of the protease undergo conformational changes > 0.15 A upon binding of the peptides; namely, those segments comprising binding sites S2 to S4. Comparison of the overall binding modes of the three tetrapeptides in the complexes indicates that the peptide aldehyde (IV) moves in a concerted manner towards His57 and Ser195, due to the formation of the covalent hemiacetal bond; the overall root-mean-square co-ordinate difference in the 33 atoms common to I and IV is 0.34 A. A very large conformational movement of the imidazole ring of His57 (χ1 changing by 101 ° and χ2 by 7 °) is observed in the aldehyde complex. Of 200 water molecules located within the first contact shell of the enzyme, only four are internally bound, two of which are structurally equivalent to internal water molecules in the pancreatic serine proteases. There is a chain of water molecules ordered approximately parallel to the polypeptide chain forming the S1 to S3 binding sites. Sixteen water molecules which occupy the active site vicinity (Sielecki et al., 1979) are displaced by the product and inhibitor molecules when they are bound to the enzyme. The results from this study provide evidence for some important modifications to the reaction pathway of serine proteases, from formation of the initial E · S complex to the final dissociation of the E · P complex. We consider that the main motive forces for conversion of the enzyme-substrate complex to the covalent tetrahedral intermediate are the electrostatic interactions of the peptide dipole moments of the oxyanion binding site. We propose that the acyl enzyme is a high energy ester with a pyramidal carbonyl carbon atom, the carbonyl oxygen atom remaining in the strongly polarizing electrostatic field of the oxyanion site. The electronic strain accumulated in the acyl enzyme is released in the formation of the tetrahedral product intermediate. The most stable intermediate in this reaction sequence at pH 4.1 is the enzyme-product complex, which we have isolated in two cases (I and II). The close contact from Oγ of Ser195 to the carbonyl carbon atom of the product indicates that the energy barrier between the tetrahedral product and the planar carboxylate product intermediates must be relatively small, and is consistent with a partial bond between these two atoms.

Refined structure of α-lytic protease at 1.7 Å resolution analysis of hydrogen bonding and solvent structure

The structure of alpha-lytic protease, a serine protease produced by the bacterium Lysobacter enzymogenes, has been refined at 1.7 A resolution. The conventional R-factor is 0.131 for the 14,996 reflections between 8 and 1.7 A resolution with I greater than or equal to 2 sigma (I). The model consists of 1391 protein atoms, two sulfate ions and 156 water molecules. The overall root-meansquare error is estimated to be about 0.14 A. The refined structure was compared with homologous enzymes alpha-chymotrypsin and Streptomyces griseus protease A and B. A new sequence numbering was derived based on the alignment of these structures. The comparison showed that the greatest structural homology is around the active site residues Asp102, His57 and Ser195, and that basic folding pathways are maintained despite chemical changes in the hydrophobic cores. The hydrogen bonds in the structure were tabulated and the distances and angles of interaction are similar to those found in small molecules. The analysis also revealed the presence of close intraresidue interactions. There are only a few direct intermolecular hydrogen bonds. Most intermolecular interactions involve bridging solvent molecules. The structural importance of hydrogen bonds involving the side-chain of Asx residues is discussed. All the negatively charged groups have a counterion nearby, while the excess positively charged groups are exposed to the solvent. One of the sulfate ions is located near the active site, whereas the other is close to the N terminus. Of the 156 water molecules, only seven are not involved in a hydrogen bond. Six of these have polar groups nearby, while the remaining one is in very weak density. There are nine internal water molecules, consisting of two monomers, two dimers and one trimer. No significant second shell of solvent is observed.

Protein structure refinement: Streptomyces griseus serine protease A at 1.8 A resolution.

Abstract The crystal structure of the bacterial serine protease from Streptomyces griseus (SGPA) has been refined at 1.8 A resolution by a restrained parameter least-squares procedure ( Konnert, 1976 ) to a conventional R factor of 0.139 for 12662 statistically significant reflections [ I > 3 σ ( I )]. The number of variable parameters in the final model was 5912 which included positional and individual thermal parameters of the enzyme, and positions, B factors and occupancies of 175 solvent molecules. The algorithm used for this refinement allows for the simultaneous restraint on bond distances and distances related to interbond angles, the coplanarity of atoms in planar groups, the conservation of chirality of asymmetric centres, non-bonded contact distances, conformational torsional angles and individual isotropic temperature factors. The refined structure of SGPA differs from ideal bond lengths by an overall root-mean-square deviation of 0.02 A; the corresponding value for angle distances is 0.038 A. Comparison of the phase angles for the shell of data, 8.0 to 2.8 A, between the multiple isomorphous replacement phases (Brayer et al. , 1978 a ) and the refined phases, indicates an overall difference (r.m.s.) of 56.6 °. The average conformational angle of the peptide bond (ω) is 179.7 ° (root-mean-square deviation ± 2.5 °) for the 180 peptide bonds of SGPA. Of the 175 solvent molecules included during the course of the refinement, 22 with occupancies ranging from 1.00 to 0.38 are located in the active site and the substrate binding region. It was not until these water molecules were included in the refinement process that the active Ser195 adopted its final conformation ( χ 1 = −77 °). The resulting distance from O γ of Ser195 to N e 2 of His57 is 3.1 A, which, when taken with the observed distortion from linearity (50 °), indicates a rather weak interaction.

Mechanism of acid protease catalysis based on the crystal structure of penicillopepsin.

A proposed mechanism for the catalytic hydrolysis of peptide bonds by acid proteases is similar in many respects to the Zn–carbonyl mechanism previously derived for carboxypeptidase A. In the acid proteases the electrophilic component is the proton shared by Asp-32 and Asp-215; Tyr-75 donates its proton to the amide nitrogen of the scissile bond and an OH− ion from a water molecule bound between the carboxyl group of Asp-32 and the substrate attacks the carbonyl carbon atom.

Molecular structure of the α-lytic protease from Myxobacter 495 at 2·8 Å resolution

Abstract The α-lytic protease was isolated from an extracellular filtrate of the soil microorganism Myxobacter 495. Trigonal crystals (space group, P 3 2 21) of this serine enzyme were grown from 1·3 m -Li 2 SO 4 at pH 7·2. X-ray reflections from crystals of the native enzyme, comprising the 2·8 A limiting sphere, were phased by the multiple isomorphous replacement technique. Five heavy-atom derivatives were used and the overall mean figure of merit 〈 m 〉 is 0·83. The resulting native electron density map of α-lytic protease has been interpreted in conjunction with the published sequence (Olson et al. , 1970) of 198 amino-acid residues. α-Lytic protease has a structural core similar to that of the pancreatic serine proteases (108 α-carbon atom positions are topologically equivalent (within 2·0 A) to residues of porcine elastase) and its tertiary structure is even more closely related to the two other bacterial serine protease structures previously determined (James et al. , 1978; Brayer et al. , 1978b; Delbaere et al. , 1979a). α-Lytic protease has the following distinctive features in common with the bacterial serine enzymes, Streptomyces griseus proteases A and B: an amino terminus that is exposed to solvent on the enzyme surface, a considerably shortened uranyl loop (residues 65 to 84), a major segment of polypeptide chain from the autolysis loop deleted (residues 144 to 155), a buried guanidinium group of Arg138 in an ion-pair bond with Asp194, and an altered conformation of the methionine loop (residues 168 to 182) relative to the pancreatic enzymes. At the present resolution, the members of the catalytic quartet (Ser214, Asp102, His57 and Ser195) adopt the conformation found in all members of the Gly-Asp-Ser-Gly-Gly serine protease family. The carboxylate of Asp102 is in a highly polar environment, as it is the recipient of four hydrogen bonds. The interaction between the N e 2 atom of the imidazole ring in His57 and O γ atom of Ser195 is very weak (3·3 A) and supports the concept that there is little, if any, enhanced nucleophilicity of the side-chain of Ser195 in the native enzyme. The molecular basis for the observed substrate specificity of α-lytic protease is clear from the distribution of amino acid side-chains in the neighborhood of the active site. An insertion of five residues at position 217, and the conformation of the side-chain of Met192 account for the fact that the specificity pocket can bind only small residues, such as Ala, Ser or Val.

Molecular structure of crystalline Streptomyces griseus protease A at 2.8 å resolution: II. Molecular conformation, comparison with α-chymotrypsin and active-site geometry☆

The present 2.8 A resolution structure determination of the microbial enzyme Streptomyces griseus protease A (SGPA) reveals a molecular architecture similar to that of the pancreatic serine proteases. The major structural dissimilarities between SGPA and the pancreatic enzymes are associated with the functional differences of these regions. Limited proteolytic cleavage of the zymogen precursors of the pancreatic proteases results in the formation of an ion-pair from the newly-formed amino-terminus to Asp194, and thereby in the active conformation of the enzymes. Inactive zymogen precursors for SGPA or other related bacterial serine proteases have not been reported. This is consistent with the present observations that the critical ion-pair involving the carboxylate group of Asp194 is formed permanently with the buried guanidinium group of Arg138. Although sequence homology between the bacterial serine proteases and the pancreatic enzymes is minimal (< 21%), the topological equivalence of the α-carbon atoms of SGPA and α-chymotrypsin is substantial (~60%, James et al., 1978). The residues in close proximity to the active site are most similar, not only in sequence but also in tertiary structure. In fact, we find almost identical conformations for the four residues of the active site (Ser214, Asp102, His57 and Ser195) to those found in the pancreatic enzymes. Although the carboxylate of Asp102 is buried, it is located in a highly polar environment as this group is the recipient of four hydrogen bonds. This fact indicates that the pKa of this group, were it directly measurable, would be considerably lower than expected for a freely available carboxylate moiety. The environment of Asp102 in SGPA is also similar to the environment of Asp32 of the acid protease penicillopepsin (Hsu et al., 1977) and the pKa of this latter carboxyl group is ~1.5. The Oγ of Ser195 is not in a position to form an ideal hydrogen bond to Ne2 of His57. The conformation of the seryl side-chain is the “down” position (χ1 ~ −80°) and we find no evidence for a conformation in which χ1 is different by ~180° from this value, as has been reported for native α-chymotrypsin (Birktoft & Blow, 1972). The overall degree of similarity of structure between SGPA and α-chymotrypsin leaves little doubt that these two enzyme families have a common ancestral origin.

Crystal structure studies and inhibition kinetics of tripeptide chloromethyl ketone inhibitors with Streptomyces griseus protease B.

Abstract The bacterial serine protease, SGPB, was inhibited by two specific tripeptide chloromethyl ketones, N-t-butyloxycarbonyl- l -alanylglycyl- l -phenylalanine chloromethyl ketone (BocAGFCK) and N-t-butyloxycarbonyl-glycyl- l -leucyl- l -phenylalanine chloromethyl ketone (BocGLFCK). Crystals of the inhibited complexes were grown and examined by X-ray crystallographic methods. The peptide backbone of each inhibitor is bound by three hydrogen bonds to the main chain of residues Ser214 to Gly216. There are two well-characterized hydrophobic pockets, S1 and S2, on the surface of SGPB which accommodate the P1 and P2 side-chains of the BocGLFCK inhibitor. A conformational change of Tyr171 is induced by the binding of this inhibitor. Both inhibitors make two covalent bonds to the SGPB enzyme. The imidazole ring of His57 is alkylated at the Nϵ2 atom and Oγ of Ser195 forms a hemiketal bond with the carbonyl-carbon atom of the inhibitor. Comparison of the binding modes of the two tripeptides in conjunction with the differences in their inhibition constants (KI) allows one to estimate the binding energy of the leucyl side-chain as −2.6 kcal mol−1. The importance of an electrophilic component in the serine protease mechanism, which involves the polarization of the susceptible carbonyl bond of a substrate or inhibitor by the peptide NH groups of Gly193 and Ser195 is discussed.

Molecular structure of crystalline Streptomyces griseus protease A at 2.8 Å resolution: I. Crystallization, data collection and structural analysis

Excellent tetragonal crystals of the A protease from Streptomyces griseus were grown by equilibrium dialysis from 1.3 m-NaH2PO4 at pH 4.1. There are four molecules in the unit cell (axial lengths a = b = 55.14(4) A, and c = 54.81(3) A); the space group is P42. Intensity data were collected on a Picker FACS-1 diffractometer to minimum d spacings of 2.8 A for crystals of the native enzyme and four heavy-atom derivatives. Background corrections to the measured peak intensities were made by a least-squares fit to a multi-dimensional function of the net intensity (I) and a linear combination of 2θ and φ. There was no dependence of the background on χ. The phase determination process has resulted in an overall average flgure-of-merit of 0.82 for 3957 reflections. The overall ratio of the root-mean-square (r.m.s.) heavy-atom scattering factor to the r.m.s. lack-of-closure errors for the derivatives ranged from 1.53 to 3.64. The common sodium mersalyl and mercury chloranilate site was close to the imidazole ring of His57; the rhenium site was located in a pocket between two enzyme molecules. The r.m.s. deviation of the measured co-ordinates to a stereochemically fitted model was 0.25 A for the 1265 non-hydrogen atoms of the A protease.

Penicillopepsin: 2.8 a Structure, Active Site Conformation and Mechanistic Implications

The crystal structure of penicillopepsin, an extracellular acid protease isolated from the mold Penicillium janthinellum, has been determined at 2.8 A resolution by the method of multiple isomorphous replacement. The resulting electron density map computed from the native structure factor amplitudes and MIR phases has an overall mean figure of merit of 0.90. The molecule is decidedly nonspherical, with the majority of residues in beta-structure. There is an 18-stranded mixed beta-sheet which forms the structural core in the region of the active site. This site, identified by the covalent binding of two EPNP molecules to Asp-32 and Asp-215, is located in a deep groove which divides the molecule into two approximately equal lobes. Both aspartic acid residues in the active site are in intimate contact with one another and the carboxyl group of Asp-32 makes two other important hydrogen-bonded contacts: one with Ser-35 and the other with the main chain peptide bond between Thr-216 and Gly-217. A proposed mechanism for acid protease catalysis is similar in many aspects to that proposed for carboxypeptidase A. The electrophilic component which polarizes the substrate carbonyl bond in the acid proteases is the proton shared between the beta-carboxyl groups of Asp-32 and Asp-215. The beta-carboxyl group of Asp-32 removes a proton from a water molecule bound between this side chain and the substrate; the resultant OH- attacks the carbonyl carbon atom of the substrate molecule. The phenolic -OH group of Tyr-75 donates its proton to the amide nitrogen of the scissile bond of the substrate.

Structure of the complex formed between the bacterial-produced inhibitor chymostatin and the serine enzyme Streptomyces griseus protease A☆

Abstract Chymostatin is a naturally occurring inhibitor of serine proteases that have chymotryptic-like specificity. This tetrapeptide inhibitor is produced by various species of Streptomyces bacteria. Chymostatin reacts with the serine enzyme Streptomyces griseus protease A in the crystalline state to produce an adduct, the structure of which is in agreement with hemiacetal formation between the C-terminal l -phenylalaninal residue of the inhibitor and the O γ atom of the active Ser195 residue of S. griseus protease A. The 2.8 A difference electron density map of the complex is also consistent with the novel structural features previously deduced spectroscopically for chymostatin; i.e. an essential (for inhibition) aldehyde function in the C-terminal l -phenylalaninal residue, an unusual arnino acid, 2-(2-iminohexahydro-(4 S)-pyrimidyl)-(S)-glycine as the third residue from the C terminus and an N-terminal amino group blocked by a (1S)-carboxyphenylethyl-carbamoyl group. There is no significant movement of the active site residues of S. griseus protease A upon complexation with chymostatin.